Compositions and Methods for Reducing Oxidative Damage

ABSTRACT

Polymeric compositions are provided that include a poly(ethylene glycol), a viscoelastic polymer, and an antioxidant, where, in polymerized form, the compositions have a refractive index of about 1.30 to about 1.40. Methods of synthesizing the compositions are also provided and include the steps of heating an amount of water; adding a buffering agent to the water to form a buffer solution; mixing a poly(ethylene glycol) and a viscoelastic polymer into the buffer solution to form a reactive mixture; adding a plurality of antioxidant particles to the reactive mixture; and removing suspended gas bubbles from the reactive mixture. Methods of preventing oxidative damage to an eye lens of a subject are further provided and include administering the foregoing polymeric compositions to the eye lens of the subject.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/646,660, filed May 21, 2015, which is a 35 U.S.C. § 371 NationalStage Application of International Application No. PCT/US2013/071319,filed Nov. 21, 2013, which claims priority from U.S. ProvisionalApplication Ser. No. 61/728,900, filed Nov. 21, 2012, the entiredisclosure of which is incorporated herein by this reference.

TECHNICAL FIELD

The presently-disclosed subject matter relates to compositions andmethods for reducing oxidative damage. In particular, thepresently-disclosed subject matter relates to compositions and methodsfor reducing oxidative damage that make use of a combination ofpolymeric materials and an antioxidant for protecting an eye lens fromoxidative damage.

BACKGROUND

The lens of the eye is a biconvex transparent structure that helps tofocus light onto the retina, where mature fiber cells of the lenscontain high amounts of protein and are important for the transparencyand refractive power of the lens. Normally, these proteins are protectedfrom oxidation by reducing substances and by the low-oxygen environmentaround the lens. Indeed, within the eye, oxygen concentration decreasessharply from the retina towards the lens due to the presence of thevitreous gel. As such, surgical removal or involutional degeneration ofthe vitreous gel often increases the exposure of the lens to oxygenoriginating from the retinal vasculature.

Vitrectomy is the surgical method used to remove some or all of thevitreous gel from the eye. It is an essential step of many vitreoretinalsurgical procedures and is performed on approximately 500,000 patientsper year in the United States alone. However, removal of the vitreousoften results in an efflux of oxygen inside the eye through diffusionfrom the retina-choroid complex and/or ion-assisted transport of oxygenthrough the vitreous gel. In addition, oxygen is also introduced throughthe surgical incisions and gases or solutions used to temporarilyreplace the vitreous gel. As a result, nearly half of patients developsecondary cataracts within two years after undergoing a vitrectomy dueto increased intraocular oxygen levels.

Current practices after vitrectomy to reduce the risk of cataractformation include requiring patients to maintain tedious head-downpositions to keep the gas bubbles away from the lens until the bubblesare completely absorbed. Unfortunately, maintaining a head-down positionfor a prolonged time is often highly challenging, especially among theelderly, and may have several side-effects, such as ulnar nervepinching. Thus, half of the patients facing these requirements aftervitrectomy are ultimately not compliant. Nevertheless, there iscurrently no other medical or surgical method effective to prevent theonset of post-vitrectomy cataracts.

Accordingly, there remains a need for a composition and/or method thatcan maintain low oxygen pressure around the eye lens to preventoxidative damage and the formation of lens opacities, but that is alsobiocompatible, does not require invasive surgery, and does not undulycompromise a subject's vision.

SUMMARY

The presently-disclosed subject matter meets some or all of theabove-identified needs, as will become evident to those of ordinaryskill in the art after a study of information provided in this document.

This summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this summary or not. To avoid excessiverepetition, this summary does not list or suggest all possiblecombinations of such features.

The presently-disclosed subject matter includes compositions and methodsfor reducing oxidative damage. In particular, the presently-disclosedsubject matter includes compositions and methods for reducing oxidativedamage that make use of a combination of polymeric materials and anantioxidant for protecting an eye lens from oxidative damage.

In some embodiments of the presently-disclosed subject matter, apolymeric composition is provided that comprises a poly(ethyleneglycol), a viscoelastic polymer, and an antioxidant, where, inpolymerized form, the composition has a refractive index of about 1.30to about 1.40 to allow the polymeric composition to be effectively usedin the eye lens of a subject. In some embodiments, the refractive indexof the compositions is about 1.33 to about 1.36.

The poly(ethylene glycol) included in the compositions described hereincan, in some embodiments, vary depending on the intended use of thecomposition and can include poly(ethylene glycol) alone or can includepoly(ethylene glycol) linked to other functional moieties that assist inthe assembly of the polymeric composition. For example, in someembodiments, the poly(ethylene glycol) included in the compositions ispoly(ethylene glycol) diacrylate. In some embodiments, the poly(ethyleneglycol) has a molecular weight of about 2000 Da to about 20000 Da and,in some embodiments, is included in the composition at a concentrationof about 50 mg/mL to about 150 mg/mL.

With regard to the viscoelastic polymers included in the compositions,in some embodiments, the viscoelastic polymer is selected from the groupconsisting of hyaluronic acid or a salt thereof, hydroxymethylpropylcellulose, chondroitin sulfate, polyacrylamide, collagen, dextran,heparin, agarose, chitosan, and a combination thereof. In someparticular embodiments, the viscoelastic polymer is hyaluronic acid or asalt thereof, and the hyaluronic acid or the salt thereof is included inthe composition at a concentration of about 8 mg/mL to about 12 mg/mL.In other embodiments, the viscoelastic polymer is hydroxymethylpropylcellulose, and the hydroxymethylpropyl cellulose is included in thecomposition at a concentration of about 1 mg/mL to about 40 mg/mL. Insome embodiments, the hydroxymethylpropyl cellulose has a viscosity ofabout 200 cP to about 5600 cP at 2% concentration in water at 20° C.,and, in some embodiments, the hydroxymethylpropyl cellulose has amolecular weight of about 200,000 Da. Further, in some embodiments, thehydroxymethylpropyl cellulose included in the compositions can varydepending on the intended use of the composition and can includehydroxymethylpropyl cellulose linked to other functional moieties thatassist in the assembly of the polymeric compositions. For instance, insome embodiments, the hydroxymethylpropyl cellulose included in thecompositions is hydroxymethylpropyl cellulose acrylate.

Regardless of the particular visco-elastic polymer and poly(ethyleneglycol) materials included in a composition of the presently-disclosedsubject matter, once combined and polymerized, the poly(ethylene glycol)and the viscoelastic polymer generally take the form of aninter-penetrating polymer, a cross-linked polymer, or a combinationthereof. In some embodiments, to tune the properties of the compositionsand provide a composition that can effectively be utilized in the eyeand, more specifically, with the eye lens of a subject, thepoly(ethylene) glycol and the viscoelastic polymer are included in thecomposition at a ratio of poly(ethylene glycol) to viscoelastic polymerof about 5:3, about 5:2, about 5:1, about 6:1, about 7:1, about 8:1,about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1,about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, or about20:1. In some embodiments, the composition further comprises anemulsifier, a non-ionic surfactant, or both that can allow for a greatervariance in the ratio of poly(ethylene glycol) to viscoelastic polymerincluded in an exemplary polymeric composition.

Turning now to the antioxidants included in the compositions of thepresently-disclosed subject matter, in some embodiments, the antioxidantincluded in an exemplary polymeric composition is selected from thegroup consisting of trehalose, nicotinamide, ascorbic acid,N-acetylcysteine, sodium azide, pyridoxine, alpha tocopherol,tocopherol, hydrazine, glutathione, thiol, beta-carotene, lycopene,astaxanthin, thioredoxin, tocochromanol, plastoquinol, cyanine,dismutase, enzymes, catalase, divalent cations, zinc, magnesium, orcombinations thereof. In some embodiments, the antioxidant is trehalose.In some embodiments, the antioxidant is included in the composition at aconcentration of about 0.001 wt % to about 10 wt %. Further, in someembodiments, the antioxidant is included in the composition as aplurality of antioxidant particles such as, in some embodiments,antioxidant particles having a diameter of about 50 nm to about 1000 nm.

To assist in the polymerization of the components of the compositions,once combined, in some embodiments, an initiator is further included inthe compositions to initiate and/or promote polymerization of thepoly(ethylene glycol) and the viscoelastic polymer. The initiator can bea photoinitiator or an enzyme. In some embodiments, the initiator is aphotoinitiator and is selected from the group consisting of2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, eosin Y,triethanolamine, 1-vinyl-2-pyrrolidinone, and combinations thereof. Insome embodiments of the compositions that make use of a photoinitiator,the viscoelastic polymers include a photo-crosslinking moiety that iscapable of interacting with other components of the compositions (e.g.,the poly(ethylene glycol) and assisting in the polymerization of thecompositions.

To allow the compositions of the presently-disclosed subject matter tobe effectively utilized with the eye lens of a subject, in someembodiments, the components of the compositions are configured (e.g.,tuned) to impart certain characteristics or properties on the polymericcompositions. For instance, in some embodiments, the compositions areconfigured such that the composition comprises a surface energy ortension of less than about 4 dyne/cm or greater than about 40 dyne/cm,such that the adherence of proteins and cells to the compositions ismitigated. As another example, in some embodiments, the compositions areconfigured to have an elasticity of about 50 N/m to about 1000 N/m so asto reduce any potential interference with the shape of the natural eyelens of a subject. Further, in some embodiments, the composition has anosmolarity of about 281 mOsm to about 350 mOsm that allows thecomposition to be substantially isosmotic with the vitreous gel of aneye. Moreover, oxygen permeability is another property that can beadjusted in the compositions described herein and, in some embodiments,is from about 1% to about 80% in order to provide a composition thatsufficiently reduces oxidative damage to the lens, and prevents theestablishment of a perilenticular oxygen gradient. As yet anotherexample of a tunable property, in some embodiments, the composition isthermally stable between a temperature of about 33° C. to about 37° C.,such that the compositions are biocompatible and stable in the eye of asubject.

Further provided, in some embodiments of the presently-disclosed subjectmatter, are methods of synthesizing a composition such as thosedescribed herein. In some embodiments, a synthesis method is providedthat includes the steps of heating an amount of water; adding abuffering agent to the water to thereby form a buffer solution (e.g.,phosphate-buffered saline or a2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)buffering solution); mixing a poly(ethylene glycol) and a viscoelasticpolymer into the buffer solution to thereby form a reactive mixture;adding an antioxidant to the reactive mixture; and then removingsuspended gas bubbles from the reactive mixture before allowing thereaction mixture to polymerize. In some embodiments, an emulsifier, anon-ionic surfactant, or both are also added to the reactive mixture.Additionally, in certain embodiments, an initiator, such aphotoinitiator, an enzyme, or a combination thereof, is further added tothe reaction mixture to assist in and provide control over thepolymerization of the reaction mixture. For example, in someembodiments, the initiator included in the reactive mixture is aphotoinitiator, such that, after the step of removing the suspended gasbubbles, the reactive mixture is exposed to electromagnetic radiation(e.g., visible light, ultraviolet light, or combinations thereof) topolymerize the compositions.

Still further provided, in some embodiments of the presently-disclosedsubject matter, are methods of reducing oxidative damage to an eye lensof a subject. In some embodiments, a method of reducing oxidative damageto an eye lens of a subject is provided that includes first providing acomposition including a poly(ethylene glycol), a viscoelastic polymer,and an antioxidant, where the composition has a refractive index ofabout 1.30 to about 1.40 in polymerized form. Then, the composition isadministered to the eye lens of the subject. In some embodiments, thetherapeutic methods can further include the step of polymerizing thecomposition subsequent to administering the composition to the eye lensof the subject. For example, in some embodiments, the compositionfurther includes a photoinitiator, such that, once the composition isadministered to a subject, the composition and the eye of a subject canbe exposed to an amount of electromagnetic radiation and the compositioncan be polymerized within the eye of a subject. In some embodiments, thestep of administering the composition comprises injecting thecomposition through a needle (e.g., a 25 gauge to 27 gauge needle) orother applicator into the eye of a subject.

Further features and advantages of the presently-disclosed subjectmatter will become evident to those of ordinary skill in the art after astudy of the description, figures, and non-limiting examples in thisdocument.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the percent transmission of light as afunction of the wavelength of light through a polymeric compositioncomprised of a mixture of poly(ethylene glycol)-diacrylate andhyaluronic acid (PEG-DA/HA) and a polymeric composition comprised ofPEG-DA/HA including 7.5 wt % trehalose;

FIG. 2 is a graph showing the refractive index of a polymericcomposition of the presently-disclosed subject matter comprised of 100mg/mL poly(ethylene glycol)-diacrylate (PEG-DA), 10 mg/mL hyaluronicacid (HA), and 7.5 w % trehalose sub-micron particles;

FIG. 3 is a scanning electron microscopy (SEM) image of a lyophilizedpoly(ethylene glycol)-diacrylate and hyaluronic acid hydrogel inaccordance with the presently-disclosed subject matter comprised of 100mg/mL poly(ethylene glycol)-diacrylate (PEG-DA), 10 mg/mL hyaluronicacid (HA), and 7.5 w % trehalose sub-micron particles;

FIG. 4 is a SEM image of a polymeric composition of thepresently-disclosed subject matter comprised of 100 mg/mL poly(ethyleneglycol)-diacrylate (PEG-DA), 10 mg/mL hyaluronic acid (HA), and 5 w %trehalose sub-micron particles;

FIG. 5 is a higher magnification of the SEM image shown in FIG. 4, andfurther showing the trehalose particles positioned in the pores of thepolymeric composition.

FIG. 6 is a graph showing the antioxidant capacity of trehaloseparticles versus the antioxidant capacity of trehalose powder, where theantioxidant capacity of the particles and powder was measured as thepercent inhibition of oxygen radicals over two time periods;

FIG. 7 is a graph showing the variation in the refractive indexes ofpolymeric compositions of the presently-disclosed subject matter as afunction of temperature;

FIG. 8 is a graph showing changes in weight of a polymeric compositionof the presently-disclosed subject matter after soaking the polymericcomposition in water for a period of 30 days;

FIG. 9 is a graph showing the changes in weight of a polymericcomposition of the presently-disclosed subject matter over an initial120-hour period after soaking the polymeric composition in water;

FIG. 10 is a series of images of freshly-isolated porcine lenses showingthe opacification of the lenses upon culturing the lenses for a timeperiod;

FIG. 11 is a pair of images showing the formation of a cataract in aporcine lens subsequent to culturing the lens with 1 mM H₂O₂ over a timeperiod;

FIG. 12 is a series of lenses showing the formation of a cataract in aporcine lens subsequent to culturing the lens with 0.5 mM H₂O₂ over atime period;

FIG. 13 is a pair of images showing the effect of coating a porcine lenswith a polymeric composition of the presently-disclosed subject matter,where the composition failed to cause an opacity in the lens over a timeperiod;

FIG. 14 is a graph showing the effect of coating a porcine lens with apolymeric composition of the presently-disclosed subject matter onoxygen diffusion into the lens; and

FIG. 15 is a pair of images showing a reduction in opacification of aporcine lens coated with a polymeric composition of thepresently-disclosed subject matter relative to an uncoated lens, whereeach lens was cultured with 0.3 mM H₂O₂.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

While the terms used herein are believed to be well understood by one ofordinary skill in the art, the definitions set forth herein are providedto facilitate explanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a protein” includes aplurality of such proteins, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±50%, in someembodiments ±40%, in some embodiments ±30%, in some embodiments ±20%, insome embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%,in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed method.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

Vitrectomies, or the surgical removal of some or all of the vitreous gelfrom the eye, are routinely performed to treat a number of eyedisorders, including diabetic eye disease. Despite the usefulness ofvitrectomies in treating those disorders, however, the removal of thevitreous gel also routinely results in the formation of cataracts,either during the early post-operative period or during the latepost-operative period. In particular, during the early post-operativeperiod, prolonged contact of gas bubbles with the posterior lens surfaceresults in the lens surface being exposed to an increased amount ofoxygen that, in turn, causes in damage to the lens. During the latepost-operative period, that exposure to oxygen then further takes theform of oxidative stress, which only serves to further damage the lensof the eye. In this regard, the attenuation of oxidative stress isthought to potentially provide a means to reduce cataract formation andlens opacity following a vitrectomy, and many antioxidant-containingcompositions such as intraoperative irrigation solution, topical drops,and oral antioxidants have been developed to attempt to combat theoxidative stress that follows a vitrectomy. Nevertheless, thefunctionality of those prior compositions has been limited by the factthat the prior compositions had an effectiveness that was short induration (e.g., minutes) or were entirely ineffective in preventingcataracts and lens opacities.

To that end, the presently-disclosed subject matter is based, at leastin part, on the discovery that a polymeric composition can be producedthat has controlled oxygen permeability and can function as an oxygenbarrier, but that is also biocompatible, such that it can be insertedinto the eye of a subject and can offer a noninvasive option forreducing oxidative damage to the eye lens after a vitrectomy. In someembodiments, the presently-disclosed subject matter thus includescompositions and methods for reducing oxidative damage. In particular,the presently-disclosed subject matter includes compositions and methodsfor reducing oxidative damage that make use of a combination ofpolymeric materials and an antioxidant for protecting an eye lens fromoxidative damage. In some embodiments, a polymeric composition isprovided that comprises a poly(ethylene glycol), a viscoelastic polymer,and an antioxidant. In this regard and as described further below, insome embodiments, the composition is a gel or, in other words, asemi-solid substance that exhibits viscous and/or elastic properties,and that is configured to cover an eye lens and reduce oxidative damageto the eye lens.

The type and amounts of poly(ethylene glycol) selected for use in thecompositions of the presently-disclosed subject matter can, in someembodiments, vary depending on the intended use of the composition andcan be tuned to impart desired properties on the composition. In someembodiments, the poly(ethylene glycol) has a molecular weight of about2000 Da, about 3000 Da, about 4000 Da, about 5000 Da, about 6000 Da,about 7000 Da, about 8000 Da, about 9000 Da, about 10000 Da, about 11000Da, about 12000 Da, about 13000 Da, about 14000 Da, about 15000 Da,about 16000 Da, about 17000 Da, about 18000 Da, about 19000 Da, or about20000 Da, such that a poly(ethylene glycol) having a particularmolecular weight can be selected so as to ensure sufficient accumulationof the viscoelastic polymer (e.g., polysaccharide) in the polymericcomposition, to ensure that the composition has low protein adhesion,and/or to ensure that the composition is sufficiently water-soluble andbiocompatible, as described in further detail below. In someembodiments, the poly(ethylene glycol) concentrations included in thecompositions are kept low so that poly(ethylene glycol) is usedefficiently in the synthesis of the polymeric composition. In someembodiments, the poly(ethylene glycol) is included in an exemplarypolymeric composition at a concentration of about 50 mg/mL, about 60mg/mL, about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL,about 110 mg/mL, about 120 mg/mL, about 130 mg/mL, about 140 mg/mL, orabout 150 mg/mL.

Furthermore, in some embodiments of the polymeric compositions, thepoly(ethylene glycol) further comprises acrylate moieties, such as, forexample, the acrylate moiety that is included in poly(ethyleneglycol)-diacrylate (PEG-DA), and which assists in the assembly of thecomplete polymeric composition. In this regard, the term “poly(ethyleneglycol)” is thus used to refer to poly(ethylene glycol) molecules alone,but is further inclusive of poly(ethylene glycol) materials havingadditional functional groups, such as PEG-DA and the like.

With respect to the viscoelastic polymers included in the compositionsof the presently-disclosed subject matter, the term “viscoelasticpolymer” is used herein refer to a polymer that is capable of impartingviscoelastic properties on the composition, where the term“viscoelastic” generally refers to a substance that exhibits bothviscous and elastic properties. In some embodiments, the term“viscoelastic polymer” thus refers to a substance (e.g., apolysaccharide) that forms a viscoelastic composition when it reactswith poly(ethylene glycol).

Similar to the poly(ethylene glycol) portion of the polymericcompositions, in some embodiments, the types of viscoelastic polymersincluded in the compositions can also vary depending on the types andamount poly(ethylene glycol) that are used in a particular compositionand/or the intended use of a particular polymeric composition. In someembodiments, the viscoelastic polymer is a polysaccharide. In somespecific embodiments, the viscoelastic polymer is selected fromhyaluronic acid or a salt thereof, hydroxymethylpropyl cellulose,chondroitin sulfate, polyacrylamide, collagen, dextran, heparin,agarose, chitosan, or combinations thereof, as such viscoelasticpolymers have been found to impart sufficient viscoelasticity on acomposition of the presently-disclosed subject matter. Typically,however, the viscoelastic polymer is selected so that when it is mixedwith other components, such as the poly(ethylene glycol), it creates acomposition having one phase and is optically pure.

In addition to selecting a particular type of visco-elastic polymer fora particular application, in some embodiments, the concentration oramounts of viscoelastic polymer included in an exemplary composition canalso be varied to impart desired properties on an exemplary polymericcomposition. For instance, in some embodiments, hyaluronic acid isincluded in an exemplary polymeric composition as the viscoelasticpolymer at a concentration of about 5 mg/mL, about 6 mg/mL, about 7mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 11 mg/mL, orabout 12 mg/mL, about 13 mg/mL, about 14 mg/mL, or about 15 mg/mL. Insome embodiments, the viscoelastic polymer is hydroxymethylpropylcellulose, which, in some embodiments, is included in an exemplarypolymeric composition at a concentration of about 1 mg/mL, about 5mg/mL, about 10 mg/mL, about 15 mg/mL, 20 mg/mL, about 25 mg/mL, about30 mg/mL, about 35 mg/mL, or about 40 mg/mL. In other embodiments, theviscoelastic polymer includes hydroxymethylpropyl cellulose having aviscosity of about 200 cP to 5600 cP, when measured at a 2%concentration in water at room temperature (i.e., 20° C.), and, in someembodiments, the hydroxymethylpropyl cellulose has a molecular weight ofabout 200,000 Da. In further embodiments, exemplary polymericcompositions can include other viscoelastic polymers including, but notlimited to, chondroitin sulfate at a concentration of about 25 to about40%, polyacrylamine at a concentration of about 0.5% to about 25%,collagen at a concentration of about 1% to about 3%, agarose at aconcentration of about 0.5% to about 50/%, or chitosan at aconcentration of about 0.5% to about 10%.

In some embodiments of the polymeric compositions, to further enhancethe ability of the viscoelastic polymer described herein to beincorporated into and effectively used in a polymeric composition of thepresently-disclosed subject matter, the viscoelastic polymers can becombined with and/or included with certain functional moieties (e.g., aphoto-crosslinking moiety) that are capable of interacting with theother components of the composition. For example, in some embodiments,the hydroxymethylpropyl cellulose included in an exemplary compositioncan vary depending on the intended use of the composition and caninclude hydroxymethylpropyl cellulose linked to other functionalmoieties that assist in the assembly of the polymeric compositions. Forinstance, in some embodiments, the hydroxymethylpropyl celluloseincluded in the compositions is hydroxymethylpropyl cellulose acrylate.In this regard, in some embodiments, the viscoelastic polymers areprovided with moieties that are configured to interact with thepoly(ethylene glycol) portions of the compositions and allow theviscoelastic polymers to bond and polymerize with the poly(ethyleneglycol) component so as to form a gel having poly(ethylene glycol) and aviscoelastic polymer that are covalently cross-linked. In otherembodiments, the viscoelastic polymer and/or poly(ethylene glycol) donot include such functional moieties, and the resulting gel is aninterpenetrating network of the respective components, rather than across-linked gel. In some embodiments, depending on the amount and typesof cross-linking moieties including in a composition, the resulting gelcan include both cross-linked portions and interpenetrating polymerportions.

Regardless of the types and amounts of the poly(ethylene glycol) andvisco-elastic polymer included in a polymeric composition of thepresently-disclosed subject matter, in some embodiments, to further tunethe properties of the compositions and provide a composition that caneffectively be utilized in the eye and, more specifically, with the eyelens of a subject, the poly(ethylene glycol) and the visco-elasticpolymer are included in the composition at a ratio of poly(ethyleneglycol) to viscoelastic polymer is about 5:3, about 5:2, about 5:1,about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1,about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1,about 18:1, about 19:1, or about 20:1. For instance, in certainembodiments that make use of hyaluronic acid as a viscoelastic polymer,the ratio of poly(ethylene glycol) to hyaluronic acid is preferablybetween about 5:1 to about 15:1, and is more preferably about 10:1. Insome embodiments that make use of hydroxymethylpropyl cellulose as aviscoelastic polymer, the ratio of poly(ethylene glycol) tohydroxymethylpropyl cellulose is about 5:3 to about 15:3, and is morepreferably about 10:3. Furthermore, in some embodiments comprisingagarose, chitosan, and/or collagen as a viscoelastic polymer, the ratioof poly(ethylene glycol) to viscoelastic polymer is preferably about 5:1to about 15:1. Additionally, in some embodiments comprising chondroitinsulfate and/or polyacrylamide as a viscoelastic polymer, thecompositions comprise a ratio of poly(ethylene glycol) to viscoelasticpolymer of preferably about 5:3 to about 15:3. In some embodiments, toallow for a greater variance in the ratio of poly(ethylene glycol) toviscoelastic polymer, while still allowing the compositions of thepresently-disclosed subject matter to exhibit certain desired propertiesin their final polymerized form, the compositions further include anemulsifier or non-ionic surfactant. In some embodiments, the emulsifieror non-ionic surfactant is selected from the polysorbate family,including polysorbate 20, polysorbate 40, or polysorbate 80.

Turning now to the antioxidants included in the compositions of thepresently disclosed subject matter, the term “antioxidant” is usedherein to refer to substances capable of inhibiting oxidation ofmolecules or, in other words, substances capable of inhibiting thetransfer of electrons or hydrogen from a particular substance to anoxidizing agent. In some embodiments, the term “antioxidant” can thus beused interchangeably with the term “oxygen quenching substance.” Anon-limiting list of potential antioxidants that may be used in thecompositions of the presently-disclosed subject matter include sodiumazide, pyridoxine, tocopherols, hydrazines, glutathione, thiols,beta-carotene, lycopene and astaxanthin, thioredoxin, tocochromanols,plastoquinol, cyanine dyes, enzymes such as superoxide dismutase orcatalase, or the divalent cations of zinc or magnesium. In someembodiments, the polymeric compositions described herein can comprisemore traditional antioxidants such as, in some embodiments, trehalose,nicotinamide, ascorbic acid, N-acetylcysteine, sodium azide, pyridoxine,alpha tocopherol, tocopherol, hydrazine, glutathione, thiol,beta-carotene, lycopene, astaxanthin, thioredoxin, tocochromanol,plastoquinol, cyanine, dismutase, enzymes, catalase, divalent cations,zinc, magnesium, and the like. In some embodiments, the antioxidantincluded in the polymeric compositions is trehalose.

As will be appreciated by those of skill in the art, incorporating anantioxidant into a polymeric composition of the presently-disclosedsubject matter functions to enhance the composition's ability to act asa barrier for oxygen and to neutralize reactive oxygen species. It hasbeen determined, however, that including excessive amount of anantioxidant in an exemplary composition interferes with thepolymerization of the compositions by quenching free radicalpolymerization processes. As such, in some embodiments, the compositionsof the presently-disclosed subject matter comprise about 0.001 wt %,about 0.5, wt %, about 1.0 wt %, about 1.5 wt %, about 2.0 wt %, about2.5 wt %, about 3.0 wt %, about 3.5 wt %, about 4.0 wt %, about 4.5 wt%, about 5.0 wt %, about 5.5 wt %, about 6.0 wt %, about 7.0 wt %, about7.5 wt %, about 8.0 wt %, about 8.5 wt %, about 9.0 wt %, about 9.5 wt%, or about 10.0 wt % of an antioxidant.

In some embodiments of the presently-disclosed subject matter, theantioxidants are included in the compositions in powder form,particulate form, or combinations thereof. In some embodiments, theantioxidant can thus be homogeneously mixed throughout the polymericcomposition so as to maximize the antioxidant or oxygen quenching effectof the composition without unduly affecting light transmission andliquid diffusion though the composition. For instance, in certainembodiments, the compositions can comprise antioxidant (e.g., trehalose)particles having a diameter of about 50 nm, about 100 nm, about 150 nm,about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm,about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm,about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm,about 950 nm, or about 1000 nm, such that the particles can fill poresin the composition, and can therefore provide an additional benefit offurther reducing oxygen and other gas diffusion through the composition.In this regard, in some embodiments, the antioxidants are selected suchthat the substances do not compromise the physical and opticalattributes of the compositions.

To assist in the polymerization of the components of the compositions,once combined, in some embodiments, an initiator is further included inthe composition to initiate and promote the polymerization of thepoly(ethylene glycol) and the viscoelastic polymer. In some embodiments,the initiator is a photoinitiator, wherein the photoinitiator caninitiate the polymerization of the composition upon exposure toelectromagnetic radiation having a certain wavelength, such visiblelight, ultraviolet light, or a combination thereof. In otherembodiments, the initiator is an enzyme that can initiatepolymerization. For example, in some embodiments, the initiator isglucose oxidase that mediates a redox chain initiation ofpolymerization. In some embodiments, a minimum amount of initiator isadded to polymerize the composition so as not to unduly interfere withthe physical properties of the compositions described in more detailbelow.

In some embodiments that make use of a photoinitiator for promotingpolymerization of the polymeric compositions, the photoinitiator isselected from the group consisting of2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure® 2959,CIBA-GEIGY Corporation, Tarrytown, N.Y.), eosin Y, triethanolamine(TEA), 1-vinyl-2-pyrrolidinone (NVP), or a combination thereof. Incertain embodiments, the composition comprises only2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone at a concentrationof about 0.05 to about 0.1 w/v %. In other embodiments, the compositionmakes use of a solution of about 100 mg/mL of2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone and 70% ethanol inwater and, in some embodiments, about 7 μL to about 14 μL of the2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone and 70% ethanolsolution is then added per milliliter of composition. In furtherembodiments, the initiator comprises a mixture of eosin Y,triethanolamine (TEA), and N-vinyl pyrrolidinone (NVP), and in specificembodiments, may comprise 0.01 mM eosin Y, 0.1% to 1.5% TEA, and 37 nMNVP.

As described above, to allow the polymeric compositions of thepresently-disclosed subject matter to be effectively utilized with theeye lens of a subject, in some embodiments, the respective componentsare selected and proportioned to achieve certain characteristics in theresulting composition. More specifically, in certain embodiments, theconcentration and type of poly (ethylene glycol) as well as theconcentration and type of viscoelastic polymer are adjusted to achievecertain characteristics in the resulting composition. For instance, insome embodiments, the amount and type of the components included in anexemplary composition are selected so that the resulting composition isbiocompatible and stable in the eye, particularly, in some embodiments,at temperatures between about 33° C. to about 37° C. In someembodiments, the compositions are configured such that, when placed onthe eye lens, the compositions remain stable for a time period of about6 months or longer.

Further, in some embodiments, the compositions are also configured tohave a particular viscosity, elasticity, oxygen permeability,osmolarity, and resistance to protein and cell adhesion. In someembodiments, an exemplary composition has a viscosity within a rangethat permits the composition to be injected through a needle, including,in some embodiments, 25 or 27 gauge needles. In this regard, in someembodiments, the compositions are also configured to be sufficientlyelastic so as to not interfere with the accommodative ability of the eyelens. In some embodiments, for example, the polymeric compositionsdescribed herein have an elasticity of about 50 N/m to about 1000 N/m.

Oxygen permeability is another characteristic that can be adjusted inthe compositions described herein. The oxygen permeability of someembodiments can be about 1%, 2%, 3%, 4%, or 5%, about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%. Insome embodiments, the oxygen permeability is from about 0.1% to about10%, such that a composition is provided having a low oxygenpermeability that, in turn, increases the composition's ability toprevent oxidative damage to the eye lens. In some embodiments, byproviding such a composition, a barrier can thus be provided that isrelatively oxygen impermeable, such that the composition can be used toreduce the formation of a perilenticular oxygen gradient from buildingup and potentially causing oxidative damage that can lead to oraccelerate the formation of cataracts. Of course, in some embodiments,the permeability of the membrane, including the oxygen permeability, canbe readily adjusted such that the compositions are permeable to certainfluids and ions, and do not block necessary substances from reaching theeye lens. In this regard, in some embodiments, the compositions arefurther configured to have an osmolarity of about 281 mOsm to about 350mOsm to allow the compositions to be substantially isosmotic to vitreoushumor of an eye.

In addition to the configurations described above, in some embodiments,the polymeric compositions are also configured to have surface tensionsthat prevent or mitigate proteins and cells from adhering to thecomposition. This characteristic, among other things, helps to ensurethat the composition remains optically clear and does not pose long-termside effects when implanted on or applied to an eye lens of a subject.In some embodiments, the surface tension of the composition is less thanabout 4 dyne/cm. In some embodiments, the surface tension of thecomposition is greater than about 40 dyne/cm. In some embodiments,removal or decreasing the amount of poly(ethylene glycol) present in aparticular composition leads to decreased biocompatibility due toincreased protein adsorption and surface tension in the compositions.

The properties of each of the compositions of the presently-disclosedsubject matter can readily be selected for a particular application andfine-tuned by varying the types and amounts of the components of thecompositions and then testing for the desired properties using methodsknown to those skilled in the art. Typically, however, in eachembodiment of the compositions, the components are selected so that theresulting composition is optically clear, does not interrupt lighttransmission, and does not cause glare or cause a loss of contrast. Inthis regard, in each embodiment, the compositions will generally exhibitgreater than 95% transmission of light in the visible region (400-700nm) upon polymerization. Additionally, in each embodiment, thecompositions are configured such that, in polymerized form, thecompositions have a refractive index that is similar to or the same thatfound in the eye lens of a subject, and thus, does not cause excessive,if any, refractive errors when it is applied to the eye lens. In thisregard, in some embodiments, the compositions have a refractive index ofabout 1.30 to about 1.40, and preferably between about 1.30 and 1.60,and more preferably between about 1.33 and 1.36.

Of course, the various properties of the presently-describedcompositions can also be adjusted to best match the particularcharacteristics of an eye lens found in a particular subject. Forinstance, the elasticity and refractive index can be selected tocorrespond to those of the eye lens of a particular so that the eyelens' function is not unduly compromised when the present composition isapplied thereto. The components can also be selected to adjust thenon-quantitative characteristics of the composition. For instance, insome embodiments, the components are selected and combined such that theresulting composition can be spread smoothly, is cohesive and does notdetach from the eye lens, and can be leveled into a desired shape. Inthis regard, by tuning the characteristics of the present composition tomeet certain parameters, the composition can be placed on an eye lens ina minimally invasive manner, such as by injection, yet still provide astable, biocompatible composition that is then able to prevent or reduceoxidative damage to an eye lens.

Further provided by the presently-disclosed subject matter are methodsfor synthesizing the compositions described herein. In some embodiments,a method is provided where an amount of water (e.g., ultra-purede-ionized water) is first provided and heated to a boil so to removeany carbon dioxide that may be present in the water and act as abuffering system that may interfere with the final transparency of thecompositions. After boiling, a buffering agent is then added to thewater to thereby form a buffer solution (e.g., phosphate-buffered salineor a 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES).The poly(ethylene glycol) and the viscoelastic polymer are then mixedinto the buffer solution to form a reactive mixture. An initiator, ifnecessary or desired, and the desired type and amount of antioxidant isthen subsequently added to the reactive mixture. In some embodiments,the antioxidant can be provided in the form of particles by first spraydrying a solution of the antioxidant particles (e.g., by using a BuchiB-90 nano spray dryer, Buchi Corporation, New Castle, Del.) to producesub-micron diameter (e.g., 100-200 nm) particles prior to introducingthem into the reaction mixture. After inserting the various componentsinto the reaction mixture, the mixture is then mixed and, to removesuspended gas bubbles that may compromise the physical or opticalcharacteristics of the composition and may potentially expose the eyelens to oxygen, centrifuged for a period of time sufficient to removethe gas bubbles from the composition.

In some embodiments of the synthesis methods described herein, thereactive mixture is then allowed to react to form the gel or, in otherwords, a polymeric composition of the presently-disclosed subjectmatter. As described above, however, in some embodiments that make useof an initiator that provides the reactive mixture with the ability topolymerize, the initiator is then activated so as to initiate andpromote the polymerization of the compounds. For example, in someembodiments that make use of a photo-initiator, after removing the gasbubbles from the compositions, the reactive mixture can be exposed toelectromagnetic radiation (e.g., visible light, ultraviolet light, andcombinations thereof) to polymerize the compositions.

Still further provided by the presently-disclosed subject matter aremethods for reducing oxidative damage to an eye lens of a subject. Insome embodiments, a method of reducing oxidative damage is provided thatcomprises the steps of providing a composition of thepresently-disclosed subject matter and then administering thecomposition to the eye lens of a subject. In some embodiments, thecomposition is administered to a subject post-vitrectomy to reduce theoxidative damage to the eye lens that may otherwise be experienced bythe at subject and that may potentially lead to the development ofcataracts. Of course, those of skill in the art will appreciate that thepresent composition can also be placed on any tissue or surfacerequiring an oxygen barrier and can be used to reduce an amount ofoxidative damage on those tissues or surfaces.

The terms “reduce,” “reducing,” or “reduction” when used herein inreference to oxidative damage are used to refer to any decrease orsuppression in the amount or rate of oxidative damage to the tissue of asubject, such as the eye lens. Of course, it is understood that thedegree of reduction need not be absolute (i.e., the degree of inhibitionneed not be a complete prevention of oxidative damage) and thatintermediate levels of a reduction in oxidative damage are contemplatedby the presently-disclosed subject matter. As such, in some embodiments,the reduction in oxidative damage can be about 5%, about 10%/o, about15%, about 20%, about 25%, about 300/%, about 35%, about 400/%, about45%, about 500/%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, or about 99%.

For administration of a composition as disclosed herein, in someembodiments, the presently-described polymeric compositions can beadministered to a subject using a variety of different applicatorsincluding needles, plastic, ceramic, or metal applicators, and the like,and can be administered in unpolymerized form (e.g., as a viscousliquid), in a partially-polymerized form, or in a fully polymerizedform. For example, in some embodiments, an exemplary composition isadministered to a subject by directly injecting the unpolymerizedcomposition through a small bore needle, such as a 25 or 27 gaugeneedle, into the eye of a subject adjacent to or onto the posterior sideof the eye lens. In some embodiments, the composition can then bemanipulated by spreading or leveling the composition once it has beenapplied to a surface, and, if a photoinitiator was included, can then bepolymerized by exposing the composition and the eye to electromagneticradiation. As another example, in some embodiments, the composition canbe administered by first molding and polymerizing the composition into adesired shape, and then surgically placing the molded compositiondirectly on the eye lens. As yet another example, in some embodiments,an exemplary composition can also be administered to a subject by usinga small amount unpolymerized gel to fuse or adhere to a pre-formed,polymerized or partially-polymerized gel to the eye lens of a subject.

Regardless of the particular mode of administration used in accordancewith the methods of the presently-disclosed subject matter, thepolymeric compositions described herein are typically administered in anamount effective to achieve the desired response (i.e., a reduction inoxidative damage). As such, the term “effective amount” is used hereinto refer to an amount of the therapeutic composition (e.g., a polymericcomposition) sufficient to produce a measurable biological response(e.g., a reduction in oxidative damage). Actual dosage levels of activeingredients in a therapeutic composition of the presently-disclosedsubject matter (e.g., the antioxidants) can be varied so as toadminister an amount of the polymeric composition that is effective toachieve the desired therapeutic response for a particular subject and/orapplication. The selected dosage level and amount of the antioxidant andthe other components of the polymeric composition will depend upon avariety of factors including the activity of the antioxidant,formulation, the route of administration, combination with other drugsor treatments, severity of the condition being treated, and the physicalcondition and prior medical history of the subject being treated.Preferably, a minimal dose is administered, and dose is escalated in theabsence of dose-limiting toxicity to a minimally effective amount.Determination and adjustment of a therapeutically effective dose, aswell as evaluation of when and how to make such adjustments, are knownto those of ordinary skill in the art of medicine.

In addition to being capable of use in a method of reducing oxidativedamage in an eye of a subject, in some embodiments, the compositionsdescribed herein can further be used to provide other therapeuticbenefits. For example, because the refractive index of thepresently-described polymeric compositions are similar to thecrystalline lens of a subject, in some embodiments, the posteriorsurface of the polymeric composition can be sculpted upon polymerizationto change the refractive power of the existing lens of the subject. Inthis regard, and considering that 25% of the US population has myopiawith 30% of that affected population having high (greater than −6D)myopia, the physicochemical properties of the gel can be used to provideenhanced refractive properties to that population. In this regard, andwithout wishing to be bound by any particular theory or mechanism, it isfurther believed that doing so is advantageous as the administration ofthe polymeric composition will place it closer to the optical nodalpoint, thus allowing for a better and more physiological correction ofthe myopia, for an improved maintenance of the accommodative property ofthe lens, and for the avoidance of ocular complications of piggybackintraocular lenses and the like.

As another example of the therapeutic use of the polymeric compositionsdescribed herein, in some embodiments, the polymeric composition canfurther be combined with one or more additional therapeutic agents suchthat the compositions can be used to deliver the therapeutic agentsdirectly into the intraocular media in a slow release manner. In thisregard, and considering that the compositions are typically configuredto be administered intraoperatively during vitrectomy surgery, routinepostoperative regimens of therapeutic agents (e.g., steroids, NSAIDS,cycloplegics, and antibiotics) can be incorporated into the compositionsand delivered postoperatively along with the compositions. Of course,the therapeutic agents that can be incorporated into such a polymericcomposition are not limited to such postoperative agents, but can alsoinclude therapeutic agents such as anti-VEGF drugs for retinal vasculardiseases and/or anti-tumor, anti-fibrotic, anti-inflammatory agents,immunomodulators, encapsulated cells, and/or genetic materials that areuseful in treating a number of diseases or disorders in a subject.

As used herein, the term “subject” is inclusive of both human and animalsubjects. Thus, veterinary uses are provided in accordance with thepresently disclosed subject matter and the presently-disclosed subjectmatter provides methods for preventing oxidative damage in mammals suchas humans, as well as those mammals of importance due to beingendangered, such as Siberian tigers; of economic importance, such asanimals raised on farms for consumption by humans; and/or animals ofsocial importance to humans, such as animals kept as pets or in zoos.Examples of such animals include but are not limited to: carnivores suchas cats and dogs; swine, including pigs, hogs, and wild boars; ruminantsand/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats,bison, and camels; and horses. Also provided is the treatment of birds,including the treatment of those kinds of birds that are endangeredand/or kept in zoos, as well as fowl, and more particularly domesticatedfowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guineafowl, and the like, as they are also of economic importance to humans.Thus, also provided is the treatment of livestock, including, but notlimited to, domesticated swine, ruminants, ungulates, horses (includingrace horses), poultry, and the like.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples.

EXAMPLES Example 1—Synthesis of Oxygen Barrier Composition

To synthesize an exemplary oxygen barrier composition, in someembodiments, 1 mL of the composition is prepared by initially preparinga buffer solution by boiling 100 mL of ultra-pure de-ionized water (18MΩ resistance) for one-half hour and then adding2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES, freeacid version) to the de-ionized water to a final concentration of 10 mM.The pH of that solution is then adjusted to 7.2 using a minimal amountof sodium hydroxide. A separate eosin Y solution is then prepared bydissolving 6.4 μL eosin Y in 10 mL of ultra-pure de-ionized water (18 MΩresistance) by sonication in a bath sonicator for 30 minutes. At thesame time, trehalose particles having a sub-micron size are prepared bymixing 1 g of trehalose and 0.025 w/v° % Tween 20 in ultra-purede-ionized water (18 MO resistance). That mixture was then spray driedusing a Buchi B-90 nanospray dryer (BÜCHI Labortechnik, Flawil,Switzerland) with a 5.5 μm mesh at 80° C., 100 L/min airflow, a fastflow rate, and one-hundred percent spray capacity until all of thesolution was sprayed. The resulting trehalose particles were thenremoved from the collection drum with a Teflon® (E. I. Du Pont DeNemours and Company, Wilmington, De) spatula, and were be stored at −20°C. until use.

Upon preparation of each of the foregoing solutions and particles, theoxygen barrier composition was then synthesized by adding 100 mg/mL ofpolyethylene glycol-diacrylate (PEG-DA) and 10 mg/mL of hyaluronic acidto 1 mL of the HEPES buffer and stirring for 1 hour using an overheadstirrer. N-vinyl pyrrolidinone (3.5 All/ml), the eosin Y solution (10μl/ml), and triethanolamine (8.9 μl/ml) were then added to the mixtureof PEG-DA and the mixture was mixed with a spatula for 1 minute. 7.5 w/v% of the trehalose particles were then added and the mixture was againmixed with a spatula for 1 minute. Bubbles were then removed from themixture by centrifuging the mixture for 1 to 2 min at 300 RPM. Themixture was then placed in an single well of a well plate, and wasexposed to a LED light source emitting light having a wavelength of 520nm and an energy of about 50 mW/cm² for 60 to 90 seconds. At that time,the polymerization was sufficiently complete and formed an oxygenbarrier composition having the following formulation: 100 mg/mLpoly(ethylene glycol)-diacrylate (PEG-DA); 10 mg/mL hyaluronic acid(HA); 7.5 w/v % trehalose sub-micron particles produced viaspray-drying; and, for purposes of photo-initiation, N-vinylpyrrolidinone (NVP, 3.5 L/ml), triethanolamine (TEA, 8.9 L/ml), andEosin Y (10 μL/ml of Eosin Y stock solution).

Example 2—Characterization and Analysis of the Oxygen BarrierComposition

To characterize and analyze the synthesized oxygen barrier compositionproduced in Example 1, and to determine whether the synthesized oxygenbarrier composition has desirable optical and physical characteristicsthat would allow it to be used to prevent oxidative damage to the lensof an eye, a number of measurements were taken. From those measurements,the components of the composition could then be varied and adjusted, ifnecessary, to produce an oxygen barrier composition having the desiredproperties (e.g., optical, biocompatibility, etc.).

In the characterization of the composition, osmolarity of the pre-gelcomposition was first assessed using 50 μL of sample in a 5004MICRO-OSMETTE™ Automatic High Sensitivity 50 μL Osmometer (PrecisionSystems, Inc.) The osmolarity was calculated as an average of 5 readings(280 mOs, 289 mOs, 312 mOs, 296 mOs, 334 mOs) from the osmometer, andwas measured to be 302.2±21.3 mOsm, which was close to or matched theosmolarity typically observed in the vitreous humor of a human eye.

The gels forming the oxygen barrier compositions absorb light due to thepresence of hyaluronic acid and trehalose and, as such, the transmissionof light through the compositions was assessed using gels formed in48-well plates and a BioTek plate reader (Winooski, Vt.) operating insingle wavelength mode. In those experiments, the transmission of lightthrough the gel exceeded 95% in light wavelengths ranging from 400-700nm (FIG. 1). The readings were further confirmed using a Cary 100UV-visible spectrometer with a film holder attachment (AgilientTechnologies, Santa Clara, Calif.), and it was observed that there wassignificant UV light absorption due to the HA and trehalose componentsof the compositions.

In addition to measuring the osmolarity and light transmission, thesurface energy of the compositions was further measured using a sessiledrop method, and was found to be approximately 1 dyne/cm as the contactangle was too low for measurement and was therefore assumed to be 0-1dyne/cm. The elasticity of the pre-gel formulation of the compositionwas also measured using a Haake Caber extensional rheometer (ThermoFisher Scientific, Inc., Waltham, Mass.) and was found to be 41.5 pa byfitting the raw data to a power model for best fit.

Refractive Index measurements of the compositions were also performedusing an Anton Paar (Ashland, Va.) Abbemat refractometer. The refractiveindexes of the photopolymerized gels were found to be in a preferredrange of 1.33-1.36, which was equivalent to the refractive index of thenatural crystalline lens (1.338-1.357) of an eye of a subject. Therefractive indexes of the gels were also observed to remain stablewithin a wide range of temperature changes between 25-40° C. (FIG. 2).

To assess and fine tune the optical purity of the compositions, a numberof experiments were further undertaken in which various bufferingsystems were assessed. Upon analysis of the results from thoseexperiments, it was observed that a “low-sodium” HEPES buffer waspreferable as buffered solutions containing increased sodium levelsresulted in cloudiness in the gels. Additionally, it was found that itwas preferable to use ultra-pure water (resistance of 18 mOhms) togenerate the HEPES buffer solution. It was further found that it waspreferable to boil the HEPES buffer for approximately 30 minutes asboiling for that amount of time allowed for the removal of gasses priorto adjusting the buffer to desired pH, a process that, in turn,minimized pH adjustments by removing dissolved carbon dioxide (CO₂) fromthe water.

Ascorbic acid is known to exist in high concentration in the aqueoushumor of the eye and, as such, further experiments were undertaken todetermine the resistance of the compositions to the high ascorbatelevels that would be found within intraocular fluids. Briefly, thegelled compositions were stored in 1.4 mM ascorbate in 37° C. for twomonths with the ascorbate solution being changed for freshly madesolution every day. During that time, the degree of yellowing wasassessed visually by placing each gel over a white background and thencomparing that gel to a control gel with no ascorbate exposure. Uponanalysis of those results, it was observed that the compositionsmaintained their clarity and optical transmission characteristics withinthe solutions containing high ascorbate (1.4 mM) for more than twomonths, indicating the compositions would also be resistant to theascorbate levels typically found in intraocular fluids.

To assess the amount of trehalose particles that could be incorporatedinto the compositions without interfering with the photo-polymerizationcapabilities of the compositions, escalating concentrations of Trehalosewere incorporated into the pre-gel compositions using the synthesisprocedures described above and photo-polymerization of each compositionwas then attempted. In these experiments, it was observed that about 6%to about 10% w/v of the antioxidant could be incorporated into thecompositions without interfering with photo-polymerization, with 7.5 w/v% being found to be the preferred concentration of trehalose that couldbe incorporated into the gel formulation to act as an antioxidantwithout interfering with photo-polymerization.

To further analyze the oxygen barrier compositions, scanning electronmicroscopy (SEM) was used. In those experiments, gels were initiallyformed as described herein above and were then lyophilized overnight toremove water. The dried gels were then affixed to a stainless steelsample pedestal using conductive carbon paint. Sample gels were thencoated with a thin (approximately 0.2 nm) coating of gold/palladiumalloy using a sputter coater, and the samples were subsequently imagedusing a Zeiss Supra 35 Field emission scanning electron microscopeoperating at 2 kV beam voltage. The resulting gels appeared to beconsistent with the structure and morphology of hydrogels with thetrehalose particles positioned in the pores of the hydrogels (FIGS.3-5).

With further respect to the trehalose particles, and without wishing tobe bound by any particular theory, it was believed that trehaloseparticles may be more amendable to use as an antioxidant as compared totrehalose in powder form because the trehalose particles were thought toprovide a slow release of the antioxidant. To further examine thisbelief, the antioxidant capacity of trehalose powder and trehaloseparticles was measured using a diphenylpicrylhydazil (DPPH) assayaccording to standard protocols. More specifically, in the assay, asample of the compositions was assessed for its ability to quench thestable organic free radical in DPPH as measured by the decay of DPPHabsorbance at 530 nm. Samples were measured after 24 hours and 48 hoursin solution and, upon observing the results, it was found that trehaloseparticles had about the same antioxidant capacity as trehalose powder,but that trehalose particles allowed for an extended response (FIG. 6).

To then assess the extent to which the ratios of PEG-DA to HA in thecompositions could be varied without affecting the compositions, ratiosof PEG-DA to HA of 50/30, 75/30, 50/40, and 75/40 were used tosynthesize oxygen barrier compositions according the above-describedsynthesis methods and the refractive indexes of each of the gels weremeasured on a Metricon 2010/M prism coupler (Metricon Corporation,Pennington, N.J.). It was found that the refractive index of the gelsdid not vary greatly upon varying the ratios of PEG-DA to HA (FIG. 7).Using similar experiments in which the various concentrations oftrehalose were tested along with various buffer solutions using ahandheld refractometer, it was also observed that the addition oftrehalose did not change the refractive index of the resulting gelcompositions (Table 1).

TABLE 1 Refractive 10:1 PEG-DA:HA Gel Index (nD) With 5% w/v trehalose(PBS) 1.34695 With 5% w/v trehalose (HEPES) 1.34655 No Trehalose (HEPES)1.34085

To further assess the gel compositions and, in particular, the abilityof the gel compositions to be used to effectively act as an oxygenbarrier, the gel composition was used to coat a dissolved oxygen probeattached to a Beckman 500 series dissolved oxygen meter (BeckmanCoulter, Inc., Brea, Calif.). Upon coating the probe and placing it intodeionized water, it was observed that the gel coating was able to reducethe dissolved oxygen being measured from 10% to 0.2% in 9 minutes time(Table 2), thus indicating that the gel compositions can effectively beused as an oxygen barrier.

TABLE 2 % O₂ Time 10.9 1323 (0 Min) 15.04 1324 (+1 Min) 2.92 1325 (+2Min) 0.104 1326 (+3 Min) 0.000 1327 (+4 Min) 0.067 1328 (+5 Min) 0.2091329 (+6 Min) 0.0565 1330 (+7 Min) 0.07 1331 (+8 Min) 0.214 1332 (+9Min)

To assess whether the compositions would be capable of administration toan eye of a subject through a needle (e.g., a 25-gauge needle typicallyused to inject solutions into an eye), the viscosity of the formulationwas measured by placing 1 mL of the gelled composition in a BrookfieldDV-II Rotational viscometer (Middleboro, Ma). The formulation viscositywas found to be in the range from 8000-12000 cP, which would allow thecomposition to be applied through a 25 G cannula and would also allowthe composition to be used with conventional vitrectomy instrumentation.

During the course of the foregoing experiments, it was observed that thecompositions generally maintained their integrity and physicochemicalproperties and were able to do so for greater than 1 year as measured bymanipulating the gel with a spatula after storing for 1 year at 4° C.After being polymerized, however, it was observed that the compositionsswell due to the absorption of the water. These observations weresubsequent to soaking the gelled compositions in water and thenmeasuring the weight of the gel at various time points. That soakingresulted in a 2.2±0.13 fold increase in gel weight (FIG. 8), with themajority of the swelling occurring within the first 24 to 48 hours (FIG.9).

As the gels were to be placed in the eyes of subjects, experiments werealso undertaken to assess the proper method for sterilization and theextent of protein adsorption of the gelled compositions. To determinethe proper method for sterilization, gamma irradiation of hyaluronicacid before reconstituting the gel was initially employed; however, suchgamma irradiation altered the physic-chemical properties of the gel andwas therefore abandoned. Gas sterilization of the HA using ethyleneoxide (EtO) gas was then assessed, with the remainder of the componentsused to make the gel being filter-sterilized using filters having a 0.2micrometer pore size. It was observed that the compositions polymerizedwith the gas-sterilized hyaluronic acid and the filter-sterilizedcomponents and that the gelled compositions generally maintained theirphysicochemical properties. A slight increase in viscosity of the HAafter EtO sterilization was measured using a viscometer, whichcorrelated to a slight reduction in polymer molecular weight via gelpermeation chromatography. However, no residual compounds or chemicalmodifications to the HA were detected from the sterilization, and thecompositions gelled readily and maintained clarity.

To assess the extent of protein adsorption on the gels, studies wereconducted by soaking the hydrogels in a 10 to 100 μg/μl BSA solution for24 to 72 hours and then checking the adsorbed BSA on the gels with BCAprotein assays. Those studies revealed that, during the swelling of thegels, proteins soaked into the gel along with water, but did notintegrate into the gels themselves, and thus, were not retained withinthe structure of the gel and did not affect the optical clarity of thegels. It was further observed that the adsorbed proteins could be soakedout of the gels over time.

Finally, cell attachment studies were further conducted with the gels toanalyze whether various cells would attach to the gels once implantedinto the eyes of a subject. In this regard, 10,000 ARPE-19 cells (i.e.,a retinal pigment epithelial cell line) and fibroblasts were seeded ongels maintained in serum containing standard culture media for thosecells for 24 hours. Cell attachment was then determined using phasecontrast microscopy and MTS assays. No cell attachment was noted at 24hours for both cell types.

Example 3—Ex Vivo Analysis

To analyze the efficacy and the biocompatibility of the compositionsproduced as described above, an ex vivo analysis was undertaken in whichthe compositions were applied to eye lenses ex vivo. In theseexperiments, harvested porcine lenses were utilized. However, becausefreshly (less than 6 hrs) harvested porcine lenses typically opacifywithin 48 hours of being cultured in HEPES buffer ex vivo (see, e.g.,FIG. 10), a revised lens culture method was developed to clear thelenses and allow them to be used to assess the clarity and otherproperties of the compositions. Briefly, in these experiments, it wasfound that M199 culture medium (Gibco® #11043-023, without Phenol red,Invitrogen, Carlsbad, Calif.) supplemented with 4% sterile-filteredporcine serum, 100 units/ml penicillin and 100 μg/ml streptomycin, andadditional 5.96 g/L HEPES was preferable. In that modified medium, thelenses initially became opaque, but subsequently cleared up within 7 to10 days under normoxic conditions. After this period, the lensesremained clear and could be used for experiments.

Upon establishing the culturing conditions for the lenses, hyperoxicconditions were created and used to test the efficacy of the gelledcompositions in protecting against oxidative damage and cataractformation. Briefly, in these experiments, two methods were used to testthe efficacy of the compositions in preventing oxidative lens damage.Initially, oxidative damage with superoxide radicals generated by theaddition of hydrogen peroxide into the lens culture medium was employed.In those initial experiments, initiation of the cataract and the extentof the cataract was found to be dependent on the concentration of H₂O₂.In particular, with 1 mM H₂O₂, lens opacity first appeared at 24 hr andturned into a total cataract by day 5 (FIG. 11), whereas with 0.5 mMH₂O₂, lens opacity started at day 3 and reached to 90% on day 14 (FIG.12), and whereas with 0.4 mM H₂O₂, lens opacity started on day 5 andcovered 60% of the lens area by day 14. As a result of theseexperiments, the preferred concentration for the experiments was foundto be 0.3 mM of H₂O₂, which started to affect lens clarity on day 9 andcaused 40% of the lens area to undergo opacification by day 14. H₂O₂concentrations below 0.3 mM did not appear to cause any lensopacification for up to 28 days of ex vivo culturing. Of course, becausethe direct bathing of the lenses with H₂O₂ generatedsuper-pharmacological amounts of free oxygen radicals that resulted inlens opacification within days, unlike in human conditions wherecataract formation takes years, each of the foregoing experiments wereconducted in a closed chamber that allowed ambient oxygen amounts to betuned in fine increments and allowed lenses to be continuously bathedwith oxygenated medium, with the dissolved oxygen being continuouslymonitored with an oxymeter.

Once the parameters for exposing porcine lenses to hyperoxic conditionswere established, before exposing lenses coated with the compositionsdescribed herein to hyperoxic conditions, the effect of coating thelenses with the compositions under normoxic conditions was firstassessed. In these experiments, the unpolymerized compositions wereplaced on the lenses, photopolymerized, and then clarity of the lenseswas assessed over a period of 8 days by comparing the coated lenses touncoated control lenses. Throughout the course of the experiment, it wasobserved that the composition (i.e., the lens coating) did not cause anyopacity in cultured porcine lenses (FIG. 13), nor did it affect does notaffect lens epithelial cell viability.

To determine the effect of coating the lenses with the composition onoxygen diffusion, oxygen levels in air and M199 media were measured witha FireSting Oxygen Probe (PyroScience, Aachen, Germany), followed bymeasuring the oxygen levels at the lens cortex of a lens coated with 1mm of the compositions described herein and in the deep cortex of thelens. Probe position was confirmed using a dissecting microscope withdigital camera. Using a FireSting Oxymeter, it was observed that coatingthe compositions with the lenses resulted in an approximately 4 folddecrease in then amount of oxygen diffusing into the lens (FIG. 14).

Lenses were then coated with the compositions and exposed to hyperoxicconditions (0.3 mM of H₂O₂). Opacification of the coated lens wasprevented over the course of the experiment (FIG. 15).

REFERENCES

Throughout this document, various references are mentioned. All suchreferences, including those listed below, are incorporated herein byreference.

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1-20. (canceled)
 21. A method of reducing or preventing oxidative damageto an eye lens of a subject, the method comprising contacting an eyelens of a subject with a composition comprising a polysaccharideantioxidant, wherein the composition has a refractive index of about 1.3to about 1.4.
 22. The method according to claim 21, wherein contactingthe eye lens of the subject comprises coating the eye lens with thecomposition.
 23. The method according to claim 21, wherein thecomposition is contacted with the eye lens during vitrectomy.
 24. Themethod according to claim 21, wherein the composition is contacted withthe eye lens in an amount sufficient to prevent post-vitrectomy cataractformation.
 25. The method according to claim 21, wherein thepolysaccharide antioxidant is trehalose.
 26. The method according toclaim 25, wherein the refractive index of the composition is from about1.33 to about 1.36.
 27. The method according to claim 26, wherein thecomposition has a surface energy of less than about 4 dyne/cm or greaterthan about 40 dyne/cm.
 28. The method according to claim 27, wherein thecomposition has an elasticity of about 50 N/m to about 1000 N/m.
 29. Themethod according to claim 28, wherein the composition has an osmolarityof about 281 mOsm to about 350 mOsm.
 30. The method according to claim25, wherein the polysaccharide antioxidant is present in the compositionat a concentration of about 0.001 wt % to about 10 wt %.
 31. The methodaccording to claim 30, wherein the composition further comprises one ormore of an emulsifier and a nonionic surfactant.
 32. The methodaccording to claim 21, wherein the composition further comprises one ormore of a poly(ethylene glycol) and a viscoelastic polymer.
 33. Themethod according to claim 32, further comprising polymerizing thecomposition.
 34. The method according to claim 32, wherein thepoly(ethylene glycol) is poly(ethylene glycol) diacrylate.
 35. Themethod according to claim 32, wherein the viscoelastic polymer isselected from the group consisting of hyaluronic acid or a salt thereof,hydroxymethylpropyl cellulose, chondroitin sulfate, polyacrylamide,collagen, dextran, heparin, agarose, chitosan, and combinations thereof.36. The method according to claim 32, wherein the composition furthercomprises a photoinitiator.
 37. The method according to claim 36,wherein the photoinitiator is selected from the group consisting of2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, eosin Y,triethanolamine, 1-vinyl-2-pyrrolidinone, and combinations thereof. 38.A composition for reducing or preventing oxidative damage to an eye lensof a subject, the comprising a polysaccharide antioxidant, wherein thecomposition has a refractive index of about 1.3 to about 1.4.